With the progress of genome structure analysis of various organisms, primary structure information of candidate proteins that are predicted to act in the cell has been accumulated. However, it should be noted that the protein information extracted from genetic information is merely information, and is not an actual substance. In fact, cells and tissues are said to translate only a part of the whole genetic information in the nucleus, and the kind thereof varies depending on the origin of cells and tissues. Moreover, it seems that the quality and quantity of the protein to be expressed change from moment to moment during the development to differentiation process. In cells, moreover, a variety of proteins interact in a complicated manner to maintain life activity of the cells. It is desired that the functional analysis of gene proceed by clarifying the mutual relationship of the proteins.
Proteome analysis is an attempt to comprehensively understand various protein-protein relationships supporting the cell functions. While a reasonable methodology meeting the object is being developed, even clarification (identification) of a group of constituent component proteins involved in certain, particular metabolic reaction accompanies many difficulties in the situation now stands. Thus, comprehensive and prompt understanding of changes in proteome, which is an assembly of highly diverse proteins, is desired.
SDS-gel electrophoresis, which is conventionally used for separation of proteins, shows high separability of molecular weights. On the other hand, isoelectric focusing that performs separation based on electric charge of proteins has overcome technical problems, but has difficulty in sample preparation. While two-dimensional electrophoresis, which is a separation technique having features of both in, combination is currently one of the most superior methods in terms of separability of proteins, it has problems in reproducibility. To overcome the problems, a method using multicolor fluorescence labeling in combination, which is what is called the DIGE method, has been developed and practiced. Although automation is difficult for this method, the difficulty in ensuring reproducibility and quantitativeness has been overcome to some extent.
On the other hand, a large-scale protein identification system has been developed wherein liquid chromatography, mass spectrometer and data analysis system are connected, and the process from separation of samples to identification of protein is consistently performed automatically online. Since this system shows extremely high sensitivity and enables extremely accurate mass measurement of even a small amount of a sample, a target protein can be often identified by measuring the mass of only 2 or 3 kinds of peptide fragments derived from the protein. Alternatively, it is also possible to directly determine the amino acid sequence of a peptide by mass spectrometry and identify the protein from the amino acid sequence.
In addition, changes in the level of protein in the cells or tissues between normal ones and pathologic ones, or the level of protein expressed in the tissues with various diseases including tissue and brain under development, or tissues with altered function due to genetic mutation provide an important tip for the elucidation of pathology. Consequently, the demand not only for the technique for identifying intracellular proteins but also the technique for protein quantification is increasing.
Conventionally, the amount of a target protein has been relatively determined by indirectly detecting the binding of an antibody that specifically binds to the target protein. In this method, the target protein needs to be identified beforehand and an antibody capable of detecting the protein needs to have been obtained.
On the other hand, a method of analyzing the abundance ratio has also been used at present, which includes producing a difference in the mass of the same protein between samples using isotopes and subjecting the protein to mass spectrometry. This method is advantageous in that an unidentified protein can be identified and quantified by a single run of the analysis. For this method, labeling reagents such as ICAT (registered trade mark) reagent, iTRAQ (registered trade mark) reagent, ICPL (registered trade mark) reagent, NBS (registered trade mark) reagent and the like are utilized (see, for example, patent document 1). Moreover, many designs have been employed as evidenced by a method including allowing a digestion enzyme to independently act on samples to be compared in two kinds of water containing light oxygen atom and heavy oxygen atom, whereby peptides having mass difference of 2 are produced since oxygen is introduced as OH of carboxylic acid newly produced, and separating them by mass spectrometry to determine the quantitative ratio, a method including culturing cells with amino acids containing a light atom and a heavy atom of C, N to constitute total proteins with the amino acids, and investigating the abundance ratio of the components of the both and the like.
Among the aforementioned labeling reagents, ICAT reagent and NBS reagent are associated with various, defects. For example, it may be difficult to perform analysis depending on kinds of proteins, since these reagents bind to amino acid residues of cysteine and tryptophan, respectively, which are small in contents in proteins. In addition, comparison is limited between two kinds of samples. In addition, iTRAQ reagent and ICPL reagent used for labeling an amino group of a lysine residue of a protein are difficult to preserve, since an amide bond is formed using unstable activated carboxylic acid. Furthermore, these four kinds of reagents are all expensive, which is also a factor limiting the use thereof.    patent document 1: JP-A-2003-107066